The Physiological And Molecular Mechanisms Of Winter Turnip Rape (Brassica Rapa L.) Responses To Cold Stress

Low temperature is one of the environmental factors that limited the distribution,growth and development of crops.Therefore,the study on cold-tolerant mechanism of crop has extensively applicated value for breeding of cold-tolerant variety.Winter turnip rape?Brassica rapa L.?is a only winter oil crop in northwestern China for its adaption to cold and dry environment and is rich in many effective cold tolerance genes.In this study,we used the cold tolerant winter turnip rape variety Longyou 7 and cold sensitive variety Tianyou 4 as experiment material.Leaf anatomic structure,physiological-biochemical index the photosynthetic responses,proteomics and microRNA sequencing and cloning of PsbR gene were determined under the condition of low temperature,which revealed the response mechanism of low temperature from the level of psychology,protein and RNA.The results were summarized as follows:1.Physiological and biochemical responses to low temperature stress in B.rapa.Stomatal traits and leaf anatomic structure were observed and showed that stomatal density,stomatal area and total stomatal circumference per unit area in two cultivars decreased.Moreover,leaf thickness,palisade tissue and sponge tissue thickness became thinner and cellular space grew bigger under low temperature.Compared with Tianyou 4,Longyou 7 had smaller stomatal density,stomatal area,total stomatal circumference per unit area and leaf thickness.The parameters of photosynthesis and fluorescence were analyzed and showed that photosynthetic rate?P_n?,maximum photochemical efficiency?F_v/F_m?,the potential activity of PS??F_v/F_o?,photosynthetic performance index?PI?,F_o?O phase?,F_k?K phase?,F_j?J phase?and F_m?P phase?of chlorophyll fluorescence induced kinetics curves of O-J-I-P as well as oxygen-evolving complex ativity state(F_o-k),Q_A reduction ability(F_k-j),Q_B slow reduction PQ activity(F_i-P)and Q_B?including fast reduction PQ?reduction ability(F_j-i)of leaves in Longyou 7 decreased significantly more than those in Tianyou 4.Further studies on the physiological and biochemical showed thatthe soluble protein content and CAT activity of Tianyou 4 were higher than those of Longyou 7 and the MDA content of the former was lower than that of the latter under cold stress.Combined with the variation tendency of photosynthetic parameters,damage to cell membranes and photosystemII as well as photosynthetic inhibition of Longyou 7 were more serious than thoese of Tianyou 4.These results indicated that leaves of‘Longyou 7'have higher sensitivity to cold stress than do those of Tianyou 4.The physiological and biochemical indexes in roots were studied and showed that EL level of Longyou 7 was significantly lower that of Tianyou 4,but other physiological indexes?soluble protein,soluble sugar and superoxide?of‘Longyou 7'were higher than those of Tianyou 4.These results indicated the roots of Longyou 7 with higher tolerance to freezing stress than those of Tianyou 4.The contents of endogenous hormones?IAA?ZR?GA₃ and ABA?and total polyphenols were sampled to analyze.The results showed that compared with Longyou 7,the contents of IAA?ZR?ABA and total polyphenols as well as ratio of?IAA+ZR+GA₃?/ABA in Tianyou 4 leaves were higher,but contents of ABA and total polyphenols in its roots were lower under low temperature.The correlation analysis showed that overwint-ering rate of winter turnip rape had negative correlation with contents of IAA?ZR and GA₃ in leaves and roots under lower temperature,but had positive correlation with contents of ABA and total polyphenols of roots.These results indicated that contents of ABA and total polyphenols are very important for cold tolerance of winter turnip rape.2.Proteomics profiling induced by low temperature in B.rapaUsing iTRAQ technology,142 differently accumulated proteins?DAPs?were identified between cold-stressed leaves of Longyou 7 and Tianyou 4,and these DAPs participated in10 pathways.174 DAPs were identified between roots of two varieties,and these DAPs participated in 8 pathways.Based on the functional analysis,we concluded that decreased energy metabolism via the TCA cycle,PPP,glyoxylate and dicarboxylate,together with decreased photosynthesis,enabled leaves of winter turnip rape to balance synthesis and consumption of sugar.This adaptive balance permitted the survival of winter turnip rape under low temperature stress.We also concluded that increased ribosome and biosynthesis of secondary metabolites,decreased energy and carbohydrate metabolism via PPP,OXPHOS,fructose and mannose metabolism could improve protein synthesis,cell wall thickness of root and accumulation of sugar,which are conducive to survival of winter turnip rape under freezing stress.3.Analysis of low temperature stress responsive microRNAs in B.rapaTwo conserved miRNAs?miR166e-3p and miR166h-3p?and four conserved miRNAs?miR166e-3p,miR319e-1,miR319a,miR319-2?were identified from the leaves and root of two low-temperature-stressed winter turnip rape varieties.These conserved miRNAs belonged to members of miR166 and miR319 families.Morever,four novel miRNAs?Bra-Novel-m3153-5p,Bra-Novel-m0894-3p,Bra-Novel-m3936-5p,Bra-Novel-m0040-3p?were identified from the root of two low-temperature-stressed winter turnip rape varieties.It was speculated that miR166,miR319 and these novel miRNAs play an important role in winter turnip rape response to cold stress.Prediction of miRNA and functional analysis were found that TCP4 and MYB104 are target genes of miR319,which involved in secondary cell wall synthesis,indicating miR319 and the target genes might respond to cold stress by regulating secondary cell wall synthesis.4.Cloning and expression analysis of PsbR gene in B.rapa under Low temperature stressThe PsbR was separated and cloned from the leaf by differential display reverse transcription PCR?DDRT-PCR?and rapid-amplification of cDNA ends?RACE?.The full length cDNA of PsbR is 570 bp and the open reading frame?ORF?encoding a protein of138 amino acids,was 417 bp.PsbR belongs to the PsbR superfamily,which presented high similarity with PsbR proteins from B.oleracea var.Oleracea,E.salsugineum and B.juncea.The relative expression of PsbR gene under low temperature stress went down.Combined with decreased net photosynthetic rate?P_n?and efficiency of primary conversion of light energy of photosystem??F_v/F_m?,we speculated the expression of the PsbR gene may be inhibited under low temperature stress,and then decreased net photosynthetic rate and photochemical efficiency of PS?.